Germ cells are the cells that give rise to the next generation of an organism. To perform this specialized task, germ cells must be totipotent and immortal. They acquire these characteristics by utilizing specialized regulatory mechanisms and factors, including germline-specific granules that overlie nuclear pores and are critical for germline development. Germ granules are cytoplasmic organelles that are conserved in germ cells from worms to mammals, and are associated with the immortal and totipotent properties of germ cells. To date, the mechanisms by which germ granules function are unknown. This proposal investigates the structure and function of germ granules in the model system C. elegans using a combination of genetic and biochemical approaches. The first aim tests whether a nuclear pore-like protein domain found within an already identified germ granule component interacts with and extends the nuclear pore environment. Nuclear pore proteins interact through this domain to form a permeability barrier between the cytoplasm and nucleus. Germ granule proteins that contain this nuclear pore-like domain will be tested to see if they interact with each other, and with nuclear pore associated components. In vivo tests will investigate these interactions in living cells. The second aim tests the hypothesis that germ granules create a microenvironment that can selectively exclude access to the nuclear pore and regulate nuclear import in the germ line. The third aim identifies new components required for proper germ granule assembly. I have already performed a genome-wide RNAi screen for such components. Classes of genes identified by this screen are beginning to reveal the mechanisms by which germ granules function. Most of these genes are conserved and so may inform us about germ granule functions across species. Our studies of germ cell biology in worms may contribute insights into regulatory mechanisms shared by germ cells, stem cells and cancer cells.